The process of healing when tissue is subjected to trauma, such as wounding or burns, is an extremely complex one, but it is known to be mediated by a number of protein factors. These factors are essential to the growth and differentiation of the cells which serve to replace the tissue destroyed. A number of candidate factors have been identified on the basis of the ability of extracts from various tissues, such as brain, pituitary, and hypothalamus, to stimulate the mitosis of cultured cells. Numerous shorthand names have been applied to the active factors in these extracts, including platelet-derived growth factor (PDGF), macrophage-derived growth factor (MDGF), epidermal growth factor (EGF), tumor angiogenesis factor (TAF), endothelial cell growth factor (ECGF), fibroblast growth factor (FGF), hypothalamus-derived growth factor (HDGF), retina-derived growth factor (RDGF), and heparin-binding growth factor (HGF). (See, for example, Hunt, T. K., J Trauma (1984) 24:S39-S49; Lobb, R. R., et al, Biochemistry (1984) 23:6295-6299).
Folkman, J., et al, Science (1983) 221:719-725, reported that one of the processes involved in wound healing, the formation of blood vessels, is profoundly affected in tumors by heparin. From this and other studies, it is clear that heparin specifically binds to protein(s) associated with a number of these growth factor activities, and therefore heparin has been used as a purification tool. It has been shown that the affinity of some growth factors for heparin is independent of overall ionic charge, since both positively and negatively charged factors are bound (Maciag, T., et al, Science (1984) 225:932-935;Shing, Y., et al, Science (1984) 223:1296-1299; Klagsbrun, M., et al, Proc Natl Acad Sci (USA) (1985) 82:805-809). The capacity to bind or not to bind to heparin is one measure of differentiation between the activities in the various extracts. For example, EGF and PDGF do not bind strongly to heparin; in fact, EGF does not bind to heparin at all. The other factors above do show strong heparin binding. However, it is believed that acidic brain FGF, ECGF, RDGF, and HGF-alpha are in fact the same factor. Similarly, it is also believed that pituitary FGF, cationic brain FGF, TAF, and HGF- are the same protein. (Lobb, R. R., et al (supra)). A summary and comparison of thirteen endothelial growth factors which have been purified using heparin affinity is found in Lobb, R., et al, J Biol Chem (1986) 261:1924-1928.
Using heparin affinity chromatography, basic fibroblast growth factors exhibiting a potent mitogenic activity for capillary endothelium have been isolated from rat chondrosarcoma (Shing, Y., et al, supra) and from bovine cartilage (Sullivan, R., et al, J Biol Chem (1985) 260:2399-2403). Thomas, K. A, et al, Proc Natl Acad Sci (USA) (1984) 81:357-361, U.S. Pat. No. 4,444,760, purified two heterogeneous forms of an acidic bovine brain fibroblast growth factor having molecular weights of 16,600 and 16,800 daltons. Gospodarowicz and collaborators have shown the presence in both bovine brains and pituitaries of basic fibroblast growth factor activities and purified these proteins using heparin-affinity chromatography in combination with other purification techniques (Bohlen, P., et al, Proc Natl Acad Sci (USA) (1984) 81:5364-5368; Gospodarowicz, D., et al (ibid) 6963-6967). These factors also have molecular weights of approximately 16 kd, as does a similar factor isolated from human placenta (Gospodarowicz, D., et al, Biochem Biophys Res Comm (1985) 128:554-562).
The complete sequence for basic FGF derived from bovine pituitary has been determined (Esch, F., et al, Proc Natl Acad Sci (USA) (1985) 82: 6507-6511). Homogeneous preparations were obtained and showed potent mitogenic activity in in vitro assays with endothelial cells (basic FGF has an ED.sub.50 of 60 pg/ml).
Acidic FGF has an ED.sub.50 of about 6,000 pg/ml. An N-terminal sequence for acidic FGF derived from bovine brain tissue was determined by Bohlen, P., et al, EMBO J (1985) 4:1951-1956. Gimenez-Gallego, G., et al, determined the N-terminal sequences for both acidic and basic FGF prepared from human brain, and compared them to the bovine sequences (Biochem Biophys Res Comm (1986) 135:541-548). Their results are consistent with those disclosed herein. Also, the complete amino acid sequence of bovine brain-derived acidic FGF was determined from the isolated protein (Gimenez-Gallego, G., et al, Science (1985) 230:1385-1388; Esch, F., et al, Biochem Biophys Res Comm (1985) 133:554-562). These two determinations are in agreement with the exception of a single amino acid. However, Esch et al later reported that their sequence is in agreement with that of Gimenez-Gallego et al. The complete amino acid sequence of human acidic FGF was deduced from the gene (Jaye, M., et al, Science (1986) 233:541-545 and the complete human protein sequence was also determined by Gimenez-Gallego, G., et al, Biochem Biophys Res Comm (1986) 138:611-617 and Harper, J. W., et al, Biochem (1986) 25:4097-4103).
The FGF proteins described above and other growth factors are clearly effective in promoting the healing of tissue subjected to trauma (see, e.g., Sporn, M. B., et al, Science (1983) 219:1329-1331; Davidson, J. M., et al, J.C.B. (1985) 100:1219-1227; Thomas, K. A., et al, Proc Natl Acad Sci (USA) (1985) 82:6409-6413). Davidson, et al, (supra) specifically discloses the efficacy of FGF in wound healing. The basic FGF native proteins have been alleged to be useful in treatment of myocardial infarction (Svet-Moldavsky, G. J., et al, Lancet (Apr. 23, 1977) 913; U.S. Pat. Nos. 4,296,100 and 4,378,347). In addition, human basic FGF has been found to increase neuronal survival and neurite extension in fetal rat hippocampal neurons (Walicke, P., et al, Proc Natl Acad Sci (USA) (1986) 83:3012-3016), suggesting that this factor may also be useful in the treatment of degenerative neurological disorders, such as Alzheimer's disease and Parkinson's disease.
The FGF proteins described above provide an effective means to promote the repair of traumatized tissue as a result of wounding, surgery, burns, fractures or neurological degeneration. However, data is accumulating regarding certain properties of these growth factors which suggests that agonists of FGF may be more therapeutically effective than the native FGF proteins for tissue repair, and in certain circumstances that FGF antagonists may also be useful therapeutically.
For example, agonists of FGF which have greater biological activity as compared to native FGF would be more desirable for use in the wound healing indications described above. In contrast, antagonists of FGF would be extremely useful in therapies where neovascularization is a dominant pathology and it would be therapeutically useful to inhibit the process of angiogenesis. Therefore, it would also be desirable to construct FGF analogs which antagonize the effects of native FGF thereby inhibiting angiogenesis.
It is considered desirable to provide modifications to the native FGF DNA sequences reported for these growth factors in order to isolate the regions of the protein responsible for the distinct biological activities or regions important in the interactions of the factor with the cellular environment. Having determined the appropriate region or site of the specific interaction, structural analogs can be created which preserve certain activities, e.g. wound healing activity, while reducing or eliminating undesirable functions, such as the sequestration of FGF in the extracellular matrix.
It would also be desirable to insure the availability of these FGF protein analogs in large quantities and in a form free from any toxic or infectious impurities. The human form of the protein is preferred, and perhaps required, for therapeutic use. Since the DNA sequences encoding the proteins for both human acidic and basic FGF have been cloned and expressed by recombinant DNA techniques, site-directed mutagenesis may be employed to produce a variety of acidic and basic FGF analogs. The invention herein provides the means whereby acidic and basic FGF analogs can be obtained in practical quantities and in pure, uncontaminated form.